The objective of this project is the research and development of suitable bioanalytical methods to: (1) establish the structure and purity of potential anti-AIDS agents and new antiviral drugs, (2) determine the physical, chemical and biochemical properties, including octanol-water partition coefficients, of these compounds and their metabolites, and (3) measure these drugs and their metabolites in biological samples to elucidate pharmacology and to determine pharmacokinetics. High-performance liquid chromatography (HPLC), capillary electrophoresis (CE) and mass spectrometry are the emphasized techniques. The Phase II nucleoside reverse transcriptase inhibitor 2'-b-fluoro-2',3'-dideoxyadeonsine (F-ddA, lodenosine) continues to be a compound of interest. The extensive preclinical and clinical pharmacological and pharmacokinetic data that we have generated is being used to refine a species-scalable physiological pharmacokinetic model for nucleoside-based prodrugs. This model is being used to investigate the effects of various physiological and biochemical processes on prodrug disposition and activation, with emphasis on gastrointestinal absorption, blood-brain-barrier penetration into the CNS, and metabolic activation. This model is also being extended to include nucleoside-based agents that are not prodrugs such as the orally active DNA methyl transferase inhibitor 2(1H)-pyrimidinone riboside (zebularine). Collaborative biochemical pharmacology and rat pharmacokinetic studies have been initiated to define the intracellular metabolism, disposition and oral bioavailability of zebularine. Preliminary results show that even though zebularine is orally active, its bioavailability is very low. Direct fluorogenic derivatization of cellular extracts in conjunction with paired-ion HPLC has been employed for the nonradiochemical determination of sub- and low picomole amounts of intracellular F-ddATP, the active metabolite of both F-ddA and F-ddI. F-ddATP can be measured in peripheral blood mononuclear cells from patients treated with F-ddA, but sufficient data is not available to correlate with observed anti-HIV activity. The development of methods using capillary electrophoresis to measure intracellular nucleotide pools and metabolites continues. Large-volume sample stacking has been quantitatively evaluated in terms of linearity, reproducibility and chromatographic resolution for the CE analysis of mononucleotide mixtures. Signal enhancements of up to 160-fold could be achieved for the analysis of nucleotides using this technique, although the sensitivity enhancement is less in biological matrices because of loss in electrophoretic resolution due to ionic strength effects. Sample preparation methods and analysis strategies to overcome this later effect are under investigation. Sample stacking has been applied to characterize the minor components of synthetic nucleotide mixtures and to profile endogenous intracellular nucleotides in cultured MOLT-4 lymphocytes. CE with sample stacking also dramatically increases the speed and sensitivity of the determination of the oligonucleotide products generated in a palindromic oligonucleotide-directed enzymatic assay being developed for the measurement of intracellular deoxy- and dideoxynucleotides in order to more fully characterize various antiretroviral therapies. Ongoing research is currently directed toward the application of CE for the determination of intracellular nucleoside drug metabolism and to interfacing CE with mass spectrometry for structural analysis. AIDS Title: The Analytical Chemistry of Anti-AIDS Agents